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Modernizing the nonhomologous end-joining repertoire:
alternative and classical NHEJ share the stage.
Ludovic Deriano, David B Roth
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Ludovic Deriano, David B Roth. Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage.. Annual Review of Genetics, Annual Reviews,
2013, 47 (1), pp.433-55. .
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Modernizing the
Nonhomologous End-Joining
Repertoire: Alternative
and Classical NHEJ Share
the Stage
Ludovic Deriano1 and David B. Roth2
1
Departments of Immunology and Genomes & Genetics, Institut Pasteur,
CNRS-URA 1961, 75015 Paris, France; email: [email protected]
2
Department of Pathology and Laboratory Medicine and Abramson Cancer Family
Research Institute, Raymond and Ruth Perelman School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania 19104; email: [email protected]
Annu. Rev. Genet. 2013. 47:433–55
Keywords
First published online as a Review in Advance on
September 11, 2013
classical nonhomologous end joining, alternative nonhomologous end
joining, DNA damage response, DNA double-strand breaks, V(D)J
recombination, genomic instability
The Annual Review of Genetics is online at
genet.annualreviews.org
This article’s doi:
10.1146/annurev-genet-110711-155540
c 2013 by Annual Reviews.
Copyright All rights reserved
Abstract
DNA double-strand breaks (DSBs) are common lesions that continually
threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including cell death. Misrepair is also fraught with danger,
especially inappropriate end-joining events, which commonly underlie
oncogenic transformation and can scramble the genome. Canonically,
cells employ two basic mechanisms to repair DSBs: homologous recombination (HR) and the classical nonhomologous end-joining pathway (cNHEJ). More recent experiments identified a highly error-prone
NHEJ pathway, termed alternative NHEJ (aNHEJ), which operates
in both cNHEJ-proficient and cNHEJ-deficient cells. aNHEJ is now
recognized to catalyze many genome rearrangements, some leading to
oncogenic transformation. Here, we review the mechanisms of cNHEJ
and aNHEJ, their interconnections with the DNA damage response
(DDR), and the mechanisms used to determine which of the three DSB
repair pathways is used to heal a particular DSB. We briefly review
recent clinical applications involving NHEJ and NHEJ inhibitors.
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INTRODUCTION
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DSB: double-strand
break
DNA double-strand breaks (DSBs), although
common, are extremely dangerous (Figure 1).
Unlike most other DNA lesions, DSBs directly
threaten genomic integrity by disrupting the
physical continuity of the chromosome. Without repair, all genetic material telomeric to the
break is lost at the next cell division. A second
particular threat posed by DSBs arises from
repair mechanisms themselves, which, if not
executed properly, possess formidable power to
wreak genomic havoc. Misrepair of DSBs can,
of course, cause localized sequence alterations
and loss of genomic material. Arguably more
dangerous, however, is inappropriate joining
of the wrong pair of DNA ends. Inappropriate
joining can generate interstitial deletions,
inversions, and chromosome translocations,
which can initiate neoplastic transformation
by a variety of mechanisms. One is through
formation of chimeric oncogenes, the classic
example being the translocation between
chromosomes 9 and 22 in chronic myeloid
leukemia (CML), forming the Philadelphia
chromosome (86) and creating the novel bcr-abl
oncogene. Indeed, studies done before cancer
genome sequencing became commonplace
identified more than 300 gene fusions in
human neoplasms, and these are believed to
account for approximately 20% of human
cancer (83). This number is likely to grow as
more cancer genomes are studied in depth.
Complex genomic rearrangements are
also initiated by joining two centromerebearing chromosome fragments together.
This initiates a cascade of persistent cycles
Nonprogrammed
Programmed
e.g., Ionizing radiation
Genotoxic drugs
Replication errors
e.g., V(D)J recombination
Class switch recombination
Meiosis
DSB
Sensing
[Ku, MRE11 complex, Parp1]
Signal transduction
[ATM]
DNA repair
[HR, cNHEJ, aNHEJ]
Responses
Cancer
Cell-cycle arrest
Apoptosis
Senescence
Cell death
DEREGULATION
GENOMIC INSTABILITY
Figure 1
Schematic of cell responses, and their potential pitfalls, to double-strand breaks (DSBs). Abbreviations:
aNHEJ, alternative nonhomologous end joining; ATM, ataxia telangiectasia mutated; cNHEJ, classical
nonhomologous end joining; HR, homologous recombination.
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of chromosome fragmentation and aberrant
rejoining (81), leading to complex deletions/
translocations/amplifications termed complicons (149). Such complex rearrangements often
accompany gross chromosome rearrangements
(149). Even more complex genome scrambling arises from a recently described process
termed chromothripsis, defined as chromosome shattering and reassembling, which
involves multiple simultaneous DSBs and
misrepair. Again, cancer-causing lesions can
emerge from such catastrophic genomic events
(47).
COMMON LESIONS WITH
MANY SOURCES
Given the dire consequences that can attend the
failure to properly repair broken DNA ends, it
is (at least from the perspective of cancer biologists) unnerving that DSBs occur quite frequently. Endogenous sources of DSBs include
fragile sites, errors in DNA metabolism (e.g.,
replication across single-strand nicks and replication fork collapse), endogenous nucleases,
programmed genome rearrangements, physical
forces, and reactive oxygen species. Numerous
DSBs of exogenous origin, both natural (e.g.,
cosmic rays, terrestrial background radiation,
certain viruses) and man-made (e.g., weapons
of mass destruction and diagnostic and therapeutic maneuvers), threaten the genome. The
latter category includes diagnostic radiographs,
radiation therapy, genotoxic drugs used for cancer therapy, and gene-therapy strategies, which
can employ nucleases to induce DSBs in a desired target sequence (27, 45) but which can also
cleave unintended (off-target) sequences (92).
An interesting estimate of the number of DSBs
attending some of these exposures follows: a
10-hour flight from Philadelphia to Paris results in 0.05 DSB per cell; a body CT scan, 0.3
DSB per cell; the Chernobyl accident, 12 DSBs
per cell (on average); external beam radiotherapy (typical single dose of 1,800–2,000 mSv),
80 DSBs per cell (32). Clearly, the integrity of
our DSB repair systems is tested on an ongoing
basis.
CHALLENGES FACED BY
DOUBLE-STRAND-BREAK
REPAIR SYSTEMS
Given the foregoing discussion, we can postulate some basic requirements for an effective
DSB repair system: (a) high sensitivity, allowing detection of a single DSB, and the ability
to do so rapidly enough to allow proper repair
before a catastrophic event occurs; (b) a great
degree of specificity—the system must detect
DSBs but not nicks, mismatches, abasic sites,
or interstrand crosslinks, which require distinct
repair systems; (c) the ability to repair breaks
with a high degree of fidelity (without too much
collateral genomic damage); (d ) the capacity to
repair a variety of different kinds of DNA ends,
including those that are not directly ligatable
(produced, for example, by certain forms of irradiation and by reactive oxygen species); and
(e) the ability to coordinate DSB repair with the
physiological state of the cell (e.g., the timing
of repair with respect to cell-cycle status).
These requirements are further complicated
by genome size, structure, and organization.
Mammalian genomes are large, requiring DSB
repair systems to possess extraordinary sensitivity (one part per billion) and to be able to
consistently detect individual molecular signals
(such sensitivity parallels another sensory system, the rod photoreceptor, which can respond
to single photons). A second challenge is posed
by genomic heterogeneity. The accessibility
of a particular DNA sequence varies widely
with genomic context and/or physical location.
Surveillance mechanisms must reliably detect
and repair all DSBs, even those in less accessible
locations, such as highly condensed chromatin
and specialized subnuclear compartments (although not necessarily with the same rapidity). A third difficulty is raised by the fact that
mammalian genomes contain a high proportion
of dispersed, highly repetitive DNA sequences.
Repair of breaks located in such sequences by
homology-directed mechanisms could lead to
genome scrambling. These considerations may
underlie the evolutionary decision to employ
multiple mechanisms for DSB repair and to
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DNA damage
response (DDR):
multiprotein complex
recruited to the DNA
break, where it
activates signaling
pathways that lead to
cell-cycle arrest and
repair or cell death
DNA-PK:
DNA-PKcs + Ku
DNA-PKcs:
DNA-dependent
protein kinase catalytic
subunit
Ku: Ku70-Ku80
heterodimer
ATM: ataxia
telangiectasia mutated
MRE11 complex (or
MRN): MRE11RAD50-NBS1
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exercise careful control over which of these is
used to heal a particular DSB at a given time, in a
given cell, and at a particular genomic location.
DOUBLE-STRAND-BREAK
SENSING AND THE DNA
DAMAGE RESPONSE
Upon sensing a DSB, cells orchestrate a rapid,
wide-ranging set of responses that affect many
aspects of cellular physiology (e.g., transcription, chromatin remodeling, cell-cycle arrest,
senescence, and apoptosis) (32). This DNA
damage response (DDR) is mediated by the
phosphoinositol-3-kinase-like protein kinases
(PIKKs) DNA-PK (DNA-PKcs + Ku), ATM
(ataxia telangiectasia mutated), and ATR (ATM
and Rad-3 related), and by PARP1 and PARP2,
which are members of the poly(ADP-ribose)
polymerase (PARP) family (32). DNA-PK and
ATM are activated by DSBs, whereas ATR
is recruited to stalled replication forks. ATM
and the MRE11 complex (consisting of three
proteins: MRE11, NBS1, and RAD50) are key
players early in DDRs, and an intricate cross
talk between these two entities appears to be
required for downstream signaling events (104)
(Figure 1). The MRE11 complex is loaded onto
DNA ends and can recruit ATM (70). Cells deficient for MRE11 complex components show
defects in ATM activation and in localization
of ATM to the sites of breakage (127).
Once activated, ATM and ATR phosphorylate a variety of mediator proteins, which amplify the damage signal by recruiting additional
proteins, including additional PIKK substrates
(32). Once bound at a DSB, ATM initiates a
signaling cascade that regulates DNA repair,
cell-cycle checkpoints, and chromatin structure. ATM and ATR regulate many cellular
processes, including replication and repair, and
the complex network of signaling events integrates the response to DSBs with the replication and metabolic status of the cell, helping to
ensure that the appropriate repair pathway is
chosen (32). Recent work has implicated these
PIKKs in the regulation of additional metabolic
pathways (32, 116).
Deriano
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Roth
One central mediator of cellular responses
is p53, which is activated by DSBs through
the kinase activities of ATM and downstream
effectors. p53 regulates many potential outcomes, including cell-cycle arrest, apoptosis,
and senescence, all of which are responses that
appear to minimize the dangers to the cell (or to
the organism as a whole) posed by genomic instability (127) (Figure 1). The DDR also modifies chromatin in the vicinity of a DSB, including phosphorylation of the histone variant
H2AX to form γ-H2AX, which localizes to a
large region (∼2 Mb in higher eukaryotes) on
either side of the break (95). This, in turn, facilitates recruitment of other factors, resulting in
assembly of large, multiprotein complexes (32),
which may play roles in damage signaling, repair, and holding the DNA ends together, minimizing opportunities for aberrant rearrangements. Activation of the ATM kinase at DSBs
also leads to chromatin relaxation, which may
be important for allowing access of the repair
machinery (151), and represses transcription in
the vicinity of the DSB (115).
THREE SPECIALIZED
MECHANISMS FOR REPAIRING
DOUBLE-STRAND BREAKS
Given the variety of situations in which DSBs
may be formed (e.g., during meiosis, during
DNA replication, and in terminally differentiated, nonreplicating cells), the presence of several specialized repair systems is not surprising.
Homologous Recombination:
Template-Directed Repair
Pioneering studies in bacteria and yeast
revealed repair pathways that use extensive
sequence identities (sequence homology) to
template repair. These mechanisms are collectively termed homologous recombination
(HR) (129). HR mechanisms are regarded as
less error prone than other DSB repair mechanisms because they employ a template to direct
repair—the sister chromatid or homolog—so
that DSBs or even gaps can be repaired
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seamlessly and without the loss of genomic
information. A key step in initiation of HR is
exonucleolytic resection, which generates long
single-stranded tails, the critical intermediates
for initiating homologous pairing (129).
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Classical Nonhomologous End
Joining: “Willy-Nilly” End Joining
By the early 1980s, HR mechanisms were well
known and were viewed as safe mechanisms
for repairing DSBs, given the requirement for
a homologous template to direct repair. This,
along with the observation that laboratory
strains of Escherichia coli lacked the capacity to
efficiently join broken DNA ends by ligation
(79), made the surprising discovery that mammalian cells efficiently join unrelated DNA
fragments together end-to-end in a “willynilly” fashion (137). This repair mechanism
became known as nonhomologous end joining
(NHEJ), although it has now been renamed
classical NHEJ (cNHEJ). cNHEJ does not require sequence homology, although very short
sequence homologies (microhomologies) of a
few nucleotides can appear at the junctions and
may help to align ends (110). Extra nucleotides
often appear at junctions and arise from a variety of mechanisms (106, 109). cNHEJ appears
to be the dominant repair pathway used in
mammalian cells and is active throughout the
cell cycle, particularly in G0/G1 (111). In the
past decade, cNHEJ components and functional end-joining ability have been discovered
in phylogenetically distinct prokaryotes, including mycobacteria and Bacillus subtilis (8,
118, 136), indicating that cNHEJ is more
evolutionarily conserved than initially realized.
Alternative Nonhomologous End
Joining: Poorly Understood, Less
Faithful, and More Dangerous
The third and most recently discovered
category of the DSB repair mechanisms goes
by a variety of names: alternative NHEJ (alt
NHEJ or aNHEJ), microhomology-mediated
end joining (MMEJ), and B (backup)-NHEJ.
aNHEJ was discovered in yeast and in mam-
malian cells at approximately the same time
as was a backup system capable of repairing
DSBs in cells with genetic deficiencies for one
(or more) of the factors critical for cNHEJ (21,
64, 73). The subsequent discovery of aNHEJ
in cNHEJ-proficient cells, as discussed below,
indicated that it is not simply a backup pathway
used by cells to allow survival in the absence of
cNHEJ.
Three features characterize aNHEJ. First,
the junctions generally reveal excessive deletions and frequent microhomologies, although
microhomologies are not invariably present.
Second, aNHEJ is much less faithful than
cNHEJ, as it commonly leads to chromosome
translocations (57, 120, 149). Third, aNHEJ,
attended by the characteristics described
above, occurs in cells deficient for cNHEJ.
The molecular events initiating aNHEJ are
poorly understood, although both PARP1
[which competes for free DNA ends with the
Ku heterodimer of cNHEJ (Figure 2), has
been implicated in DNA damage sensing (58),
and can interact with ATM (2)] and the MRE11
complex appear to play important roles (13).
Future studies should clarify whether what
we currently term aNHEJ describes a single
pathway or is a category containing multiple
mechanisms (such as, for example, a distinct
pathway that requires microhomologies).
NHEJ:
nonhomologous end
joining
Classical NHEJ
(cNHEJ): DNA end
joining using a defined
pathway requiring Ku,
DNA-PKcs, Artemis,
XRCC4, ligase IV, and
Cernunnos/XLF
Alternative NHEJ
(aNHEJ): DNA end
joining independent of
the known cNHEJ
factors. Translocation
prone with the
tendency to employ
short sequence
homologies
(microhomologies) to
direct joining
Microhomology:
short stretches (1–10
nucleotides) of DNA
sequence identity used
to guide repair by
cNHEJ and aNHEJ
MECHANISM OF REPAIR BY
CLASSICAL NONHOMOLOGOUS
END JOINING
At first glance, cNHEJ appears relatively
straightforward: It joins DNA ends by ligation,
without requiring a complicated search for an
appropriate homologous repair template. Interesting questions, however, remain. How are
ends that are not blunt or self-complementary
modified to allow ligation (e.g., ends produced
by radiation damage often bear chemical modifications that necessitate considerable processing to render them ligatable)? How are the two
ends generated by a particular DSB maintained
as a pair to prevent inappropriate joining to
other ends that may coexist (many forms of
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5'
3'
3'
5'
DSB
Alternative NHEJ
Classical NHEJ
BREAK RECOGNITION
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Ku70-80
γ-H2AX
PARP1
DNA-PK
MRE11
complex
53BP1
END BINDING/SYNAPSIS
MRE11
complex
Mobility of DNA ends?
End bridging in trans?
Additional DDR factors
XLF?
CtIP
CtIP
END PROCESSING
Artemis
End resection
0–10 nucleotide microhomologies
Additional end processing factors?
Flap removal? Gap filling?
Additional nucleases
Polymerases
XRCC4-Ligase IV-XLF
XRCC1? Ligase III? Ligase I?
LIGATION
Minimum DNA loss, repair in cis
GENOME STABILITY
DNA loss, translocations
GENOME INSTABILITY
Figure 2
Classical and alternative nonhomologous end-joining (NHEJ) pathways. Abbreviations: DDR, DNA damage response.
damage produce multiple DSBs per cell)? How
are ends specified to undergo repair by cNHEJ
instead of HR or aNHEJ? Answers to some of
these questions have emerged from numerous
studies over the past two decades (Figure 2).
Loading NHEJ Factors
onto DNA Ends
Core cNHEJ factors include Ku and the DNA
ligase complex XRCC4-ligase IV-XRCC4-like
factor (XLF; also called Cernunnos). Core
cNHEJ factors are conserved from yeast to
mammalian cells, and some prokaryotes also
possess NHEJ capability involving Ku and
DNA ligase D (an ATP-dependent DNA
ligase) (118).
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cNHEJ is initiated by the binding of the Ku
heterodimer to the broken DNA ends, creating a scaffold for the recruitment of other factors, including DNA-PKcs, XRCC4-ligase IVXLF, Artemis, and DNA polymerases. Ku is
abundant (≈400,000 molecules per cell), and
has high affinity for DNA with a variety of end
structures, including blunt ends, 5 or 3 overhangs, and covalently sealed hairpins (43). Ku70
and Ku80 form a symmetrical, heterodimeric
ring that encircles duplex DNA with little direct contact with the DNA backbone or bases
(135). Once bound to an end, Ku can translocate along the molecule, allowing multiple Ku
heterodimers to load onto linear DNA (43).
After ligation, the resulting Ku-DNA complex is extremely stable and theoretically could
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be trapped on the DNA molecule after ligation (43). Recent evidence suggests that Ku80
is removed from DNA through a ubiquitinmediated process (97). Because the DNA-Ku
complex is able to recruit many enzymes, it has
been difficult to dissect the timing of the recruitment of DNA repair factors to the DNA
ends. However, extensive evidence supports the
idea that the access is at least partially mediated
by a functional DNA-Ku-DNA-PKcs tripartite
complex, indicating that the DNA-PK complex
must form at DNA ends at an earlier stage and
that the enzymes that process and ligate join the
DNA repair reaction at a later stage.
DNA-PKcs
Ku initially interacts with the distal termini of
DNA ends (43), protecting them from aberrant end resection and from aNHEJ (see below). Once DNA-PKcs is recruited, Ku translocates inward, by about one helical turn, allowing
DNA-PKcs to contact an approximately 10-bp
region at both termini (143). Upon binding to
the Ku-DNA complex, DNA-PKcs phosphorylates substrates (see below), promoting synapsis of DNA ends and facilitating recruitment
of end processing and ligation enzymes (85).
Although it is not clear whether end bridging
through synapsis is required for full activation
of the kinase, Ku is required for targeting DNAPKcs to DNA breaks and to fully stimulate its
kinase activity (85). Kinase activity is important
for NHEJ: Eliminating the catalytic activity
of DNA-PK sensitizes cells to DSB-inducing
agents and blocks repair of recombination activating gene (RAG)-mediated DSBs generated
during variable (diversity) joining [V(D)J] recombination (85).
A number of DNA-PKcs substrates have
been identified, including Ku, Artemis,
XRCC4, ligase IV, and XLF, but phosphorylation of these proteins individually does not
seem to affect NHEJ, suggesting functional
redundancy between these numerous phosphorylation sites (85). A recent study supports
this idea; DNA-PKcs-dependent phosphorylation of XRCC4 and XLF is functionally
redundant and might function to promote
XRCC4-XLF complex dissociation (112).
Another relevant target of phosphorylation
by DNA-PKcs is autophosphorylation of the
catalytic subunit itself. Autophosphorylation
of DNA-PKcs in trans across the DSB in the
synaptic complex appears to increase the ability
of DNA end-processing enzymes and ligases
to access DNA ends, suggesting a structural
change of the holoenzyme and/or a dissociation
of DNA-PKcs after autophosphorylation (42).
The autophosphorylation of two clusters of
residues, termed ABCDE and PQR, seems to
be an important requirement for regulating
DNA end access (85). Interestingly, whereas
phosphorylation within the DNA-PKcs
ABCDE cluster promotes access to DNA ends,
phosphorylation of the PQR cluster inhibits
access (85). These results indicate that access
of processing enzymes to DNA ends is tightly
regulated. This regulation may limit unsafe
repair of DNA breaks through, for example,
homologous recombination outside the S/G2
phases of the cell cycle, or it may discourage
aNHEJ (85, 117).
RAG: recombination
activating gene
Variable (diversity)
joining [V(D)J)]
recombination:
process of somatic
recombination
initiated by the
RAG1/2 recombinase
by which the V
(variable), D
(diversity), and J
(joining) segments of
immunoglobulin genes
or T-cell receptor
genes are assembled
during the
development of
lymphocytes
End Processing
Aligned, compatible DNA termini that possess
a 5 phosphate and a 3 hydroxyl can be directly
ligated by the cNHEJ factors described above.
However, more complex DNA ends, such as
those produced by irradiation or reactive oxygen species, or the hairpin coding ends produced during V(D)J recombination, cannot be
directly joined. Therefore, cNHEJ requires additional enzymes to prepare such DNA termini for ligation. One such enzyme is Artemis,
discovered as the gene mutated in certain
radiosensitive severe combined immunodeficiency (RS-SCID) patients (84). Once the endonuclease activity of Artemis is stimulated by
DNA-PK, it carries out hairpin opening (77).
Consequently, mice deficient in either Artemis
or DNA-PKcs are defective for hairpin opening and accumulate hairpin coding ends during
V(D)J recombination (103). Interestingly, RSSCID patients and Artemis−/− mice also exhibit
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increased sensitivity to DNA-damaging agents,
and Artemis is hyperphosphorylated after ionizing radiation (34), indicating an additional
role for Artemis in NHEJ. Consistent with
these observations, the endonuclease activity of
the Artemis-DNA-PK complex seems to be required for removing single-strand DNA overhangs containing damaged nucleotides (68).
Processing enzymes, such as WRN (Werner
syndrome protein), APLF (aprataxin-andpolynucleotide kinase-like factor), the MRE11
complex, and the BLM (Bloom) helicase, may
also participate in cNHEJ (55, 129).
In addition, XRCC4 has been shown to interact with polynucleotide kinase/phosphatase
(PNKP), a bifunctional enzyme that phosphorylates 5 -OH termini and dephosphorylates
3 -phosphate termini, therefore providing the
correct chemical end groups required for DNA
ligation (29). Recent studies have demonstrated
that Ku is a 5 -deoxyribose-5-phosphate/abasic
or apurinic/apyrimidinic (5 -dRP/AP) lyase
that excises nucleotide damage near broken
ends during cNHEJ (128). This 5 -dRP/AP
lyase activity seems specific, as it is restricted
to substrates in which excision of an abasic site
is required for ligation (128). Interestingly,
in the absence of Ku, a near-terminal abasic
site is a barrier to aNHEJ, indicating that
Ku-dependent cNHEJ is uniquely able to couple 5 -dRP/AP lyase activity to joining (102).
cNHEJ is therefore uniquely effective at
coupling this end-cleaning step to joining in
cells, helping to distinguish this pathway from
aNHEJ. Among processing activities used by
cNHEJ is the extension of DNA ends by a DNA
polymerase, which fills in the gaps at or near
the site of a DSB, and template-independent
nucleotide addition during resolution of V(D)J
recombination intermediates (98). On the basis
of amino acid sequence similarity, DNA polymerases in eukaryotes have been categorized
into four classes: the A, B, X, and Y families
(25). Three members of the pol X family have
been associated with mammalian NHEJ: pol
λ, pol μ, and terminal deoxynucleotidyl transferase (TdT), which all share BRCT (BRCA1
C-terminal) domains essential for complex
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formation between the pol X member and
core cNHEJ factors at DNA ends (98). TdT
is only expressed in cells undergoing V(D)J
recombination, where it adds N nucleotides
to coding joints, thereby increasing junctional
diversity (98). Pol μ and pol λ are ubiquitously
expressed, and although vertebrate cells that
lack pol μ, pol λ, or both are not significantly
radiosensitive, they could contribute to the
quality of repair by NHEJ (98).
End Joining
The final step in cNHEJ involves the joining of
DNA ends by the DNA XRCC4-Ligase IVXLF complex. Ligase IV has an N-terminal
catalytic domain and interacts with the αhelix of XRCC4 via a region between the two
C-terminal BRCT domains (139). Binding of
XRCC4 stabilizes DNA ligase IV and stimulates its activity (22, 54). In the presence of
Ku, the DNA XRCC4-Ligase IV-XLF complex has the ability to ligate across gaps and to
ligate one of the broken strands, even when the
other strand is not ligatable (56). Interactions
of ligase IV, XRCC4, and XLF with the Ku
heterodimer have been described, but it is not
clear whether DNA-PKcs is required for the
recruitment of these proteins to the DNA ends
or whether it simply enhances complex formation and activity (78). Therefore, it is possible
that DNA-PKcs and the ligase complex may be
recruited to DNA breaks independently rather
than in a sequential manner.
In mice, deficiency in ligase IV or XRCC4
leads to embryonic lethality due to p53dependent cell death of newly differentiated
neurons (9, 48, 51). Ku-deficient mice are viable
and fertile but also have increased levels of neuronal apoptosis, are small (approximately 50%
the size of control littermates), are severely immunodeficient because of an inability to repair
V(D)J recombination–associated DSBs, and exhibit premature aging (87, 134, 148). Although
both DNA-PKcs- and Artemis-deficient mice
are also immunodeficient because of the inability of progenitor lymphocytes to join V(D)J
coding ends (103, 108), they lack the more
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severe phenotypes observed in Ku-, XRCC4-,
and ligase IV–deficient mice. These observations support the idea that proteins such as
Artemis and DNA-PKcs, which are not broadly
conserved throughout evolution, are dispensable for the repair of a large fraction of DSBs
created under physiological conditions.
XLF/Cernunnos, the most recently identified cNHEJ factor, was discovered simultaneously in patients that display growth retardation, immunodeficiency, and radiosensitivity
(23) and by a yeast two-hybrid screen for proteins that interact with XRCC4 (3). XLF stimulates ligation of noncohesive DNA ends by the
XRCC4-ligase IV complex (4, 100). XLF has
additional roles in cNHEJ. For example, XLF
is essential for gap filling by pol λ and pol μ,
suggesting that it plays a major role in aligning the two DNA ends in the repair complex
prior to ligation (4). Recent structural analysis
indicates that the XLF-XRCC4 complex could
form extended filament-like structures that facilitate DNA end bridging during cNHEJ (6,
7). These results suggest that the XLF-XRCC4
complex participates in maintaining the stability of the broken DNA ends during repair. This
stabilization function is shared by a number of
additional so-called accessory factors (see below) and seems particularly important to completion of safe repair of DNA breaks by cNHEJ,
avoiding genome scrambling misrepair events.
ROLE OF THE DNA DAMAGE
RESPONSE AND ACCESSORY
FACTORS IN CLASSICAL
NONHOMOLOGOUS END
JOINING
One key attribute of cNHEJ is its ability to keep
track of pairs of ends generated by individual
DSBs, discouraging potentially disastrous misrepair events involving ends arising at widely
separated genomic locations (Figure 2). One
obvious way to maintain identification of appropriate end pairs would be to keep them tethered
together during end processing and repair. The
RAD50/MRE11 complex can bridge DNA
ends and may perform this function (127).
Indeed, a conserved feature of the MRE11
complex is a zinc-coordinating motif in RAD50
called the RAD50 hook that enables the dimerization of chromatin-associated MRE11 complexes and could provide a flexible link between
DNA ends (35, 138). Another plausible means
for maintaining appropriate associations between end pairs arising from a single DSB is the
assembly of DDR factors over large DNA regions of chromatin on both sides of DNA breaks
to form so-called nuclear DNA repair foci.
These foci are MRE11- and ATM-dependent,
and contain histone H2AX, MDC1, 53BP1,
and NBS1 (32). DDR factor deficiencies
increase genomic instability, including, most
remarkably, the rate of unrepaired chromosomal breaks and translocations. These findings
led to the proposal that these large multiprotein complexes, although not strictly required
for joining per se, assist in tethering the DNA
ends prior to their ligation (53, 89, 145).
LESSONS FROM ANTIGEN
RECEPTOR GENE
REARRANGEMENT AND
IMMUNOGLOBULIN CLASS
SWITCHING
Studies of the repair of physiological DSBs
generated at specific sites during lymphocyte
differentiation (programmed gene rearrangements) have provided valuable insights into
NHEJ mechanisms (13, 59, 105). Programmed
rearrangements in lymphocytes fall into two
categories that employ different mechanisms
and are undertaken at different developmental
stages. Both are initiated by lymphoid-specific
mechanisms that result in DSBs, which are
then repaired by non-cell-type-specific NHEJ
processes. V(D)J recombination occurs early
in development of B and T lymphocytes and
is responsible for assembling complete antigen receptor genes from separately encoded
germ-line gene segments (Figure 3) (59, 105,
113). This process brings together V and J elements in a combinatorial fashion to generate
immunoglobulin light chains and the T-cellreceptor α and γ genes. Immunoglobulin heavy
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VDJ recombination
V segments
Class switch recombination
D segments
J segments
Cμ Cδ
Cγ3 Cγ1 Cγ2b Cγ2a Cε
Cα
IgH locus
Sμ
Sγ1
Sγ2b
Switch regions
Transcription (AID targeting)
AID (DNA lesions leading to DSBs)
RAG1/2 (DNA DSBs)
Redundancy between
RAG PCC and DDR
in DNA end
tethering/stability?
DDR
DDR
ep
RA
G1
Ar
/2
DN
air
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RSSs
CE
SE
Sγ3
DNA repair
RAG PCC shepherds
DNA ends to cNHEJ
cNHEJ/aNHEJ
(DNA repair)
cNHEJ (DNA repair)
Excised
circle
Rearranged
variable region
Rearranged
constant region
Bone marrow (TCR loci: thymus)
Excised
circle
Peripheral lymphoid tissues
Figure 3
DNA rearrangements at the IgH locus: V(D)J recombination and class switch recombination. Abbreviations: AID, activation-induced
cytidine deaminase; CE, coding end; DDR, DNA damage response; DSB, double-strand break; NHEJ, nonhomologous end joining;
PCC, postcleavage complex; RAG, recombination activating gene; RSS, recombination signal sequence; SE, signal end; TCR, T-cell
receptor.
chains and T-cell-receptor β and δ chains require the assembly of V, D, and J gene segments. Combinatorial assembly of the complete
antigen receptor genes by choosing from several possible V, D, or J gene segments provides
a fundamental mechanism for creating a diverse
repertoire of antigen-binding sites in these
receptors.
Rearrangement is initiated by the protein
products of the RAGs, RAG1 and RAG2, which
together constitute a site-specific endonuclease that introduces DSBs adjacent to conserved
recognition sites [termed recombination signal
sequences (RSSs)] (113). Cleavage at a pair of
RSSs (e.g. one adjacent to a V segment and one
adjoining a J segment) generates four broken
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DNA ends: two blunt signal ends, which terminate in the RSS, and two covalently sealed (hairpin) coding ends. These ends are then joined by
cNHEJ in a recombinant configuration, forming a coding joint (the rearranged antigen receptor gene) and a reciprocal product termed
a signal joint. Early studies established that efficient joining requires the cNHEJ machinery
(13, 59, 105).
The second lymphocyte-specific programmed DNA rearrangement process occurs
only in mature B cells and serves to swap
the DNA segment encoding the default
C-terminal immunoglobulin effector region,
which encodes the IgM isotype, for other
germ line–encoded effector regions to create
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different classes of antibodies (e.g., IgG,
IgE, IgA) with different biological properties
(Figure 3) (5, 125). This process, termed
isotype switching or immunoglobulin class
switch recombination (CSR), is also initiated
by the introduction of DSBs in specific regions,
termed switch regions, of DNA adjacent to the
effector cassettes. Unlike V(D)J recombination, these DSBs are not precisely site-specific
but can occur at a variety of locations along
the highly repetitive switch regions. They are
also generated by an indirect mechanism that
is initiated by the activation-induced cytidine
deaminase (AID) (5, 125). Pioneering studies
revealed that the DNA ends generated during
CSR are also joined by cNHEJ, although there
are interesting differences between this process
and V(D)J recombination (Figure 3) (44, 96,
122), as discussed below.
The V(D)J Recombinase: A Special
Case of End Tethering?
Deficiencies in ATM, H2AX, MDC1, the
MRE11 complex, or 53BP1 reduce CSR levels
(13). Interestingly, these factors, although
present at RAG-mediated DSBs (31), seem to
have only modest roles in V(D)J recombination.
The most striking example is 53BP1: 53BP1
is important for the repair of AID-dependent
DNA breaks, most likely by directly participating in cNHEJ, regulating DNA end resection
and long-range DNA end synapsis, and for
activation of cell-cycle checkpoints (13). Although 53BP1 is critical for CSR in the context
of V(D)J recombination, loss of 53BP1 might
be compensated by other accessory proteins. A
similar situation is observed in the case of XLF.
XLF-deficient animals, unlike other cNHEJdeficient animals, are not especially immunodeficient (72, 133). Indeed, pro-B cell lines
derived from XLF-deficient mice, although
ionizing radiation–sensitive, perform nearly
normal V(D)J recombination, leading to the
speculation that unknown lymphocyte-specific
factors/pathways might compensate for XLF
function during V(D)J recombination (72).
One such factor could be the (lymphocytespecific) V(D)J recombinase itself: The RAGpostcleavage complex holds the DNA ends
together (1, 61) and shepherds them to the
cNHEJ repair machinery (69) (Figure 3).
Could the recombinase provide a V(D)J
recombination-specific end-tethering function
that compensates for the lack of 53BP1 or XLF?
Additional evidence that supports a role for the
V(D)J recombinase in providing RAG-specific
end tethering is provided by a more detailed
analysis of the effects that deficiencies in various factors involved in DDR or cNHEJ have
on the joining of the two types of ends produced by the V(D)J recombinase: the coding
and signal ends. The RAG proteins bind much
more avidly to signal-end pairs than to codingend pairs (1, 61), perhaps reflecting the need
for coding ends to be accessible for processing
by Artemis-DNA-PKcs before ligation. With
this in mind, our RAG-tethering hypothesis
predicts that signal-joint formation should be
more resistant than coding-joint formation to
defects in other end-tethering functions. This is
indeed the case. Although capable of supporting
V(D)J recombination, lymphocytes deficient
for certain DDR factors (the MRE11 complex,
ATM, and 53BP1) accumulate unrepaired coding ends, which can lead to subsequent chromosomal deletions and translocations (59). Interestingly, disrupting the interaction between
XLF and XRCC4 also leads to impaired coding (but not signal)-joint formation, presumably reflecting end-tethering activities of the
XLF-XRCC4 complex (112) that can be partially compensated for by signal-end tethering
provided by the RAG-postcleavage complex.
The proposed tethering function provided
by the RAG-DNA end complex does not obviate the need for additional assistance. Combined deficiencies of ATM, H2AX, or 53BP1
with XLF result in a severe block in lymphocyte development with a significant defect in the
repair of RAG-mediated DSBs (75, 90, 144).
Combined deficiency of XLF with ATM also
leads to defective signal-joint formation and accumulation of signal ends, suggesting that the
absence of both cNHEJ and DDR abilities to
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Class switch
recombination
(CSR): process of
somatic recombination
initiated by the
activation-induced
cytidine deaminase
(AID) enzyme, by
which B cells change
the production of
antibodies from one
isotype to another
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tether DNA ends affects joining, even when
the ends are supported by a RAG-postcleavage
complex (91, 144).
Another example of functional redundancy
between cNHEJ and DDR factors comes
from the analysis of signal-joint formation in
DNA-PKcs- and ATM-deficient lymphocytes.
Two recent reports point to overlapping
functions of DNA-PKcs and ATM, mediated
through their kinase activities, in promoting
efficient signal-joint formation and preventing
accumulation of signal ends (52). In response
to DSBs, DNA-PKcs and ATM phosphorylate
a large number of shared substrates, including chromatin-associated factors, cNHEJ
factors, and potentially the RAG proteins.
It is therefore difficult to attribute the flaws
in signal-joint formation in this situation
to defective tethering. Nevertheless, it is
tempting to speculate that cNHEJ and DDR
[and RAG proteins, in the special case of V(D)J
recombination] collaborate in maintaining
DNA end pairs together to facilitate proper
repair and to minimize aberrant end joining
(Figure 3) (see below for further discussion).
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BIOLOGICAL CONSEQUENCES
OF CLASSICAL
NONHOMOLOGOUS END
JOINING
cNHEJ restores the physical continuity of the
chromosome, but because it generally causes
short deletions and insertions at the junctions,
repair often alters the nucleotide sequence information immediately surrounding the repair
site. This is a distinct advantage in the case
of V(D)J recombination, which seeks to generate junctional diversity that is translated into a
diverse repertoire of antigen-binding proteins.
Recent gene therapy strategies have taken advantage of the propensity of cNHEJ to make
slight modifications at the junctions to inactivate target genes cleaved by sequence-specific
nucleases (93).
The nonconservative nature of cNHEJ has
led many to refer to it as an error-prone pathway. Nevertheless, cNHEJ generally restores
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chromosome integrity without leading to chromosome rearrangements. Given that mammalian genomes contain a great deal of DNA
sequence with no known coding function, microscopic alterations produced by repair may
often be of no consequence and can be viewed
as the price the cell is willing to pay to preserve
global genomic integrity. From a genome-wide
perspective, we would argue that cNHEJ is not
particularly error prone, especially when compared with aNHEJ.
MECHANISM OF REPAIR
BY ALTERNATIVE
NONHOMOLOGOUS
END JOINING
As discussed above, evidence for an alternative
NHEJ pathway emerged from investigation
of cells deficient for cNHEJ components.
Efficient joining of extrachromosomal DNA
fragments was observed in cNHEJ-deficient
cells (64, 73), as were rare junctions formed by
V(D)J recombination at endogenous antigen
receptor loci in cNHEJ-deficient mice (12, 17,
18, 80). The structures of these junctions often
bear the features (e.g., large deletions, microhomologies, occasional insertions of large
DNA segments of unknown origin) described
above that have been taken as signatures of
aNHEJ, although none of these features is
invariably present.
The characteristics of its products imply
that aNHEJ involves enzymes promoting
end resection, proteins that take advantage
of microhomologies (presumably to stabilize
paired intermediates), nucleases capable of
removing noncompatible 5 and 3 overhangs,
and ligation. The factors involved and the
mechanism(s) underlying aNHEJ (or even
whether it represents a single pathway or multiple pathways) remain unclear. Recent work
has implicated the MRE11 complex and CtIP
in end resection that facilitates aNHEJ (37, 60,
71, 99, 140, 146), and DNA ligase III appears to
promote the ligation step, although there may
also be a role for DNA ligase I (13, 15, 119)
(Figure 2). Recent studies show that PARP1
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and DNA ligase III are important for joining
mediated by aNHEJ at telomeres lacking the
Ku heterodimer and the protection of the
full shelterin complex (114). We refer the
interested reader to a recent review for more
details (13).
Although aNHEJ was initially viewed
as merely a backup pathway present only
when cNHEJ is disabled, recent studies have
revealed that aNHEJ can be surprisingly
efficient and that it occurs in cells proficient
for cNHEJ (88). Studies using mutant RAG
proteins demonstrated that, at least with
extrachromosomal substrates, aNHEJ occurs at approximately 10% of the frequency
of cNHEJ in both cNHEJ-proficient and
cNHEJ-deficient cells (33). CSR mediated
by aNHEJ in cNHEJ-deficient cells occurs
at roughly 50% of the frequency observed in
cNHEJ-proficient contexts (121, 142).
Is aNHEJ a Distinct Repair Pathway?
Does aNHEJ operating in cNHEJ-deficient
cells simply substitute a missing cNHEJ
factor with another enzyme borrowed from
another repair pathway? According to this
view, aNHEJ is simply a substitution variant of
cNHEJ (74). This hypothesis predicts that removal of different cNHEJ factors should affect
the nature of the products generated by aNHEJ
because each aNHEJ pathway would be unique
in its means of overcoming the deficiency of a
particular cNHEJ factor. For instance, Ku deficiency might affect end resection much more
drastically than XRCC4 or ligase IV deficiency.
This is, however, not the case. Instead, both Ku
and XRCC4 deficiency lead to similar junctions
that contain large deletions and microhomologies (57). Furthermore, the substitution model
predicts that deficiency of multiple factors
should influence the nature of the aNHEJ
reaction (efficiency, structures of junctions,
etc.). Instead, immunoglobulin CSR occurs
rather efficiently in the combined absence
of Ku and ligase IV, and produces identical
junctions (14, 16). Furthermore, DNA-endshepherding-deficient RAG mutants revealed
that aNHEJ is a robust pathway even in
cNHEJ-proficient cells (33). Additional support for aNHEJ as a bona fide pathway rather
than a variant of cNHEJ is the discovery of
aNHEJ in E. coli, which lacks components
of cNHEJ, such as Ku (30), suggesting that
aNHEJ might have preceded cNHEJ in
evolution. Together, these findings provide
strong evidence that aNHEJ is a pathway in
its own right, with components (and perhaps
functions) distinct from those of cNHEJ.
If aNHEJ is a distinct pathway, does it have
a particular biological function? aNHEJ may
simply provide a fail-safe mechanism for repair
of chromosomal breaks. This would have the
advantage of preserving large swaths of the
genome between a DSB and the telomere,
which would otherwise be lost upon cell
division. The disadvantages, however, appear
significant: loss of DNA from ends because
of extensive exonucleolytic processing and
frequent chromosome translocations (120, 142,
149), indicating that these mechanisms are
truly error prone. Indeed, recent biochemical
studies indicate that end resection uncoupled
from HR occurs in extracts from mitotic cells,
raising the intriguing possibility that one
physiological function of aNHEJ may be to
heal chromosomes broken during mitosis (94).
aNHEJ is Error Prone on a
Genome-Wide Scale
cNHEJ-deficient mice (whether they lack
Ku80, XRCC4, ligase IV, DNA-PKcs, or even
Artemis) that are also deficient for p53 invariably develop pro-B cell lymphomas harboring
oncogenic chromosomal translocations involving the IgH and c-Myc loci (or N-Myc in the
case of Artemis deficiency), all of which are catalyzed by aNHEJ and harbor microhomologies
at the breakpoints (145). These observations
not only supported the existence of an errorprone aNHEJ pathway, but also gave rise to
the concept that the more high-fidelity cNHEJ
pathway acted as a tumor suppressor (38, 50,
107) by promoting faithful joining (Figure 2).
One interpretation of these results is that,
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in the presence of functional p53, cells bearing unrepaired DSBs are (mostly) eliminated;
deactivating p53 allows the cells to survive long
enough for the ends to be repaired by (presumably slower) aNHEJ.
Cultured cells deficient for either XRCC4ligase IV or Ku exhibit the same translocation
frequency and breakpoint junction characteristics, supporting a role for aNHEJ as the major
pathway to translocation formation (14, 120,
142). Analyses of chromosomal translocation
junctions in human tumors also revealed several
features, such as microhomologies and extensive end resection, and led to the suggestion that
aNHEJ forms translocations (126, 147). Indeed, most chromosome translocations in both
cNHEJ-proficient and cNHEJ-deficient cells
appear to be generated by aNHEJ (14, 120,
142). This is supported by the observation that
their frequency is reduced in the absence of
DNA ligase III (119), one of the newly identified components of the aNHEJ pathway.
Why is aNHEJ so error prone? The foregoing discussion suggests two possibilities. The
propensity of aNHEJ to generate genome rearrangements could reflect defects in stabilizing or tethering pairs of DNA ends. During repair of DSBs by cNHEJ, DNA ends are likely
maintained in close proximity by both cNHEJ
and DDR factors, therefore promoting repair
in cis. Such a role has been illustrated for Ku, as
in its absence DNA ends undergo long-range
movements within the nucleus (123). A corresponding function may be lacking in aNHEJ.
Additionally, aNHEJ appears to repair DSBs
with slower kinetics than cNHEJ in vivo (142),
increasing the chances that more than one
DSB is present concomitantly and therefore
increasing the chances of repairing the wrong
DNA ends in trans (in the case of chromosomal
translocations).
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REPAIR PATHWAY CHOICE:
A CRITICAL REGULATORY
DECISION
At least three pathways are, in principle,
available to repair a particular DSB. Indeed,
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these pathways can compete or collaborate in
repair (65, 89, 101, 120). Given that the three
pathways have quite different outcomes, one
would expect that mechanisms exist to control
the choice of repair pathway used under
certain sets of conditions. For example, HR
mechanisms predominate during S/G2, when
a sister chromatid template is available (129).
cNHEJ, in contrast, operates throughout the
cell cycle (65) (Figure 4). cNHEJ may be the
default pathway in noncycling mammalian cells
(129). In budding yeast, DNA ends are first
available to cNHEJ. As nuclease-mediated resection proceeds, long single-stranded tails are
generated that can only be joined via HR (49).
Recent work indicates that the choice between
HR and cNHEJ in replicating or G2/M phase
yeast, and in mammalian cells, is regulated by
end resection (62, 63). In S. cerevisiae, resection
of broken ends is greatly reduced in noncycling
cells, favoring cNHEJ (129).
The initial phase of end resection is limited
to fairly short stretches, with as few as 20 bp
processed (33, 37, 132), and is carried out by the
MRE11 complex and CtIP, making the ends
available for aNHEJ. Indeed, both the MRE11
complex and CtIP have been implicated in
aNHEJ (37, 60, 71, 99, 140, 146) (Figure 2;
Figure 4). In a second phase of end resection,
the BLM helicase and exonuclease 1 generate
the long single-stranded tails required to
initiate HR (129) (Figure 4). At this stage, the
long single-stranded DNA ends become poor
substrates for binding by Ku, and cells appear
to be committed to HR (129), although this
commitment might be reversed if the tails are
trimmed by nucleases.
The presence of robust aNHEJ activity in
mammalian cells suggests that mechanisms
may exist to limit use of (translocation-prone)
aNHEJ (33, 64, 120, 121, 142). Recent data
reveal a role for 53BP1 in limiting access of nucleases to DNA ends, promoting cNHEJ (19,
20, 24, 39, 40, 114) (Figure 4). This end protection depends upon phosphorylation of 53BP1
by activated ATM, promoting recruitment of
Rif1 (28, 41, 46, 150) (Figure 4). In the absence
of Rif1 (or 53BP1), DSBs generated during
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5'
3'
3'
5'
DSB
DNA break sensors
ATM
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?
BRCA
γ-H2AX
53BP1
BLM
CtIP
EXO1
MRE11
complex
Ku70-80 Rif1
Ku, 53BP1P/Rif1
End resection
RPA
MRE11 complex/
CtIP/BRCA1
EXO1/BLM
cNHEJ
Entire cell cycle
Mostly operative in G1 phase
Initial end resection
MRE11 complex/
CtIP
Long end resection
HR
S, G2 phase
End resection
Additional nucleases?
aNHEJ
Cell cycle phase?
Figure 4
End resection as a determinant of double-strand break (DSB) repair pathway choice. Abbreviations: ATM, ataxia telangiectasia
mutated; HR, homologous recombination; NHEJ, nonhomologous end joining.
immunoglobulin CSR are extensively resected
and are not repaired by cNHEJ, leading
to persistent chromosome breaks, genomic
instability, and repair by homology-based
pathways (19, 20, 38, 141). End joining of
deprotected telomeres due to the removal of
members of the shelterin complex can also
occur by aNHEJ in the absence of 53BP1, in
a process dependent on DNA ligase III and
PARP1 (114). Thus, 53BP1 appears to be
one of the factors responsible for modulating
pathway choice between aNHEJ and cNHEJ.
Additional levels of pathway choice control
appear to be incorporated in the handling of
certain physiological DSBs. For example, during V(D)J recombination, the RAG proteins
take some responsibility for restricting repair
of the broken ends to cNHEJ, limiting their
access to HR and aNHEJ (33, 69). This may
reflect a carefully orchestrated handoff from the
RAG-DNA end complex to the cNHEJ machinery. In agreement with this notion, certain
mutations in the C terminus of RAG2 destabilize the RAG-DNA end complex and allow
increased aNHEJ (33, 36, 69). This is accompanied by genomic instability and accelerated
lymphomagenesis in the absence of p53 (36),
generating tumors bearing a complex landscape
of translocations, deletions, inversions, and
duplications (82). A second example is provided
by telomeres: The shelterin complex appears
to provide telomeres with an extra level of protection against both cNHEJ and aNHEJ (114).
NONHOMOLOGOUS END
JOINING IN THE CLINIC
Intensive study of the various properties and
mechanisms of both cNHEJ and aNHEJ have
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spurred some important clinical applications.
The observation that cNHEJ often repairs
breaks with local disruption of DNA sequences
at the joining site has inspired strategies for
cNHEJ-mediated gene disruption. One application currently in clinical trials transduces T
cells from HIV-infected individuals with a zincfinger nuclease designed to cleave within the
coding region of the CCR5 gene, which encodes
a coreceptor for the HIV virus, with the resulting NHEJ creating CCR5-disrupted cells that
are resistant to infection (93).
Our knowledge of NHEJ mechanisms also
informs new therapeutic strategies against
cancer. Genomic instability is characteristic of
many cancer cells and is thought to provide
fuel for rapid evolution of subclones, which can
then be selected for invasiveness, metastatic
potential, and drug resistance (76). Recent
genomic analyses of 489 ovarian tumors have
revealed defects in HR in half of them (26),
suggesting that the progenitor cells may have
exclusively employed NHEJ to repair DSBs.
These data suggest that inhibition of cNHEJ
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GE47CH19-Deriano
(or aNHEJ) might provide a therapeutic
approach. Indeed, a DNA ligase IV inhibitor
impedes tumor progression in mouse cancer
models (124). On the basis of the role of PARP
in DNA damage sensing, PARP inhibitors
are being used in clinical trials for therapy
of cancers with lesions in BRCA1/2 (67).
Additional rationale for the use of PARP inhibitors is provided by the recent discovery that
increased aNHEJ (which, as discussed above,
might involve PARP1) in peripheral blood
lymphocytes is correlated with an increased
risk of breast cancer (66), suggesting that
these patients may be genetically predisposed
toward utilization of this (dangerous) repair
pathway. There is increasing evidence for
downregulation of cNHEJ and upregulation of
aNHEJ in a variety of human tumors (10, 11,
130, 131). Thus, aNHEJ may present a useful
therapeutic target, depending upon on what its
normal function(s), if any, might be. Continued
study of NHEJ mechanisms and regulation of
pathway choice control should provide new
insights that can be translated into the clinic.
FUTURE ISSUES
1. What are aNHEJ’s components (other than PARP1, MRE11/CtIP, and ligase III)? What
are the mechanisms of aNHEJ-mediated translocation? Is aNHEJ relevant to physiological, biological, or evolutionary processes? Is aNHEJ involved in tumor onset, progression,
and/or therapy resistance? Is aNHEJ composed of a single defined pathway or multiple
defined pathways?
2. How is the aNHEJ versus cNHEJ and HDR pathways choice regulated?
3. What are the mechanisms regulating chromosomal DSB repair in cis (intrachromosomal)
and in trans (translocational)?
4. How does DDR mechanistically and functionally contribute to DSB repair by cNHEJ
and aNHEJ? What are the detailed mechanisms of DDR-mediated synapsis?
5. Is V(D)J recombination a special case of DSB repair? How does the RAG recombinase
regulate DSB repair and pathway choice during V(D)J recombination?
6. It is important to translate the knowledge of the DNA repair mechanism into the clinic.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
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ACKNOWLEDGMENTS
We apologize to colleagues whose work could not be cited because of space limitations. L.D. is
supported by the Institut Pasteur, the CNRS, the Fondation pour la Recherche Médicale, and
the Ville de Paris, as well as by the European Research Council under the ERC starting grant
agreement number 310917. D.B.R. is supported by grants from the National Institutes of Health
(CA104588; PN2EY018244).
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Annual Review of
Genetics
Volume 47, 2013
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Causes of Genome Instability
Andrés Aguilera and Tatiana Garcı́a-Muse p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Radiation Effects on Human Heredity
Nori Nakamura, Akihiko Suyama, Asao Noda, and Yoshiaki Kodama p p p p p p p p p p p p p p p p p p p33
Dissecting Social Cell Biology and Tumors Using Drosophila Genetics
José Carlos Pastor-Pareja and Tian Xu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p51
Estimation and Partition of Heritability in Human Populations Using
Whole-Genome Analysis Methods
Anna A.E. Vinkhuyzen, Naomi R. Wray, Jian Yang, Michael E. Goddard,
and Peter M. Visscher p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p75
Detecting Natural Selection in Genomic Data
Joseph J. Vitti, Sharon R. Grossman, and Pardis C. Sabeti p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p97
Adaptive Translation as a Mechanism of Stress Response
and Adaptation
Tao Pan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 121
Organizing Principles of Mammalian Nonsense-Mediated
mRNA Decay
Maximilian Wei-Lin Popp and Lynne E. Maquat p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 139
Control of Nuclear Activities by Substrate-Selective
and Protein-Group SUMOylation
Stefan Jentsch and Ivan Psakhye p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 167
Genomic Imprinting: Insights From Plants
Mary Gehring p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 187
Regulation of Bacterial Metabolism by Small RNAs
Using Diverse Mechanisms
Maksym Bobrovskyy and Carin K. Vanderpool p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 209
Bacteria and the Aging and Longevity of Caenorhabditis elegans
Dennis H. Kim p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 233
The Genotypic View of Social Interactions in Microbial Communities
Sara Mitri and Kevin Richard Foster p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 247
SIR Proteins and the Assembly of Silent Chromatin in Budding Yeast
Stephanie Kueng, Mariano Oppikofer, and Susan M. Gasser p p p p p p p p p p p p p p p p p p p p p p p p p p p p 275
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New Gene Evolution: Little Did We Know
Manyuan Long, Nicholas W. VanKuren, Sidi Chen, Maria D. Vibranovski p p p p p p p p p p p 307
RNA Editing in Plants and Its Evolution
Mizuki Takenaka, Anja Zehrmann, Daniil Verbitskiy, Barbara Härtel,
and Axel Brennicke p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 335
Expanding Horizons: Ciliary Proteins Reach Beyond Cilia
Shiaulou Yuan and Zhaoxia Sun p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 353
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The Digestive Tract of Drosophila melanogaster
Bruno Lemaitre and Irene Miguel-Aliaga p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 377
RNase III: Genetics and Function; Structure and Mechanism
Donald L. Court, Jianhua Gan, Yu-He Liang, Gary X. Shaw, Joseph E. Tropea,
Nina Costantino, David S. Waugh, and Xinhua Ji p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 405
Modernizing the Nonhomologous End-Joining Repertoire:
Alternative and Classical NHEJ Share the Stage
Ludovic Deriano and David B. Roth p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 433
Enterococcal Sex Pheromones: Signaling, Social Behavior,
and Evolution
Gary M. Dunny p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 457
Control of Transcriptional Elongation
Hojoong Kwak and John T. Lis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 483
The Genomic and Cellular Foundations of Animal Origins
Daniel J. Richter and Nicole King p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 509
Genetic Techniques for the Archaea
Joel A. Farkas, Jonathan W. Picking, and Thomas J. Santangelo p p p p p p p p p p p p p p p p p p p p p p p 539
Initation of Meiotic Recombination: How and Where? Conservation
and Specificities Among Eukaryotes
Bernard de Massy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 563
Biology and Genetics of Prions Causing Neurodegeneration
Stanley B. Prusiner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 601
Bacterial Mg2+ Homeostasis, Transport, and Virulence
Eduardo A. Groisman, Kerry Hollands, Michelle A. Kriner, Eun-Jin Lee,
Sun-Yang Park, and Mauricio H. Pontes p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 625
Errata
An online log of corrections to Annual Review of Genetics articles may be found at
http://genet.annualreviews.org/errata.shtml
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Contents